Studies of the molecular basis of determination and differentiation in the early embryo have been restricted to whole larvae. Because of difficulties of isolation, the primary germ layers have been little studied in spite of their fundamental importance to further development. Failure, for example, to establish a normal endoderm can result in numerous defects in the adult including incomplete digestive tract and abnormalities of the liver, kidneys and other organs of endodermal origin. In the sea urchin it is possible to separate the embryonic ectoderm and endoderm at the late gastrula and pluteus stages. This request proposes to use the techniques of molecular biology to examine the patterns of gene expression in the endoderm of this organism. In Phase 1 of this project cloned DNA will be isolated and used to identify a series of genes whose expression is restricted to the embryonic endoderm. Particular attention will be payed to those genes exhibiting temporal regulation too. Using these clones as probes for Northern blots, the time course of accumulation of the mRNAs of these genes will be measured and their possible inclusion in the pool of maternal mRNAs determined. By assaying timed polysomal and nonpolysomal mRNA fractions the timing of translation of these mRNAs will be fixed. Southern blots will then be used to determine the gene copy numbers. Finally, using in-situ hybridization procedures, the specific subset of endodermal cells expressing the genes will be determined and their lineage traced. In Phase 2 of the project isolation of antibodies specific to the endoderm genes will begin. Using immunocytochemical techniques, the antibodies will be used to localize within the tissue, the sites of protein accumulation and to start to identify the specific proteins coded for by the mRNAs. The main goal of this proposal is to acquire some initial understanding of the molecular basis for the determination and differentiation of this most basic of tissue layers. A second, much longer range goal will be to determine how such close coordination of expression of the genes of the embryonic digestive system is achieved as differentiation of the endoderm proceeds.